Microfermentor and process of using



Jan. 31, 1967 R. STEEL 3,301,769

I MICROFERMENTOR AND PROCESS OF USING Filed Sept. 28, 1964 V s Sheets-Sheet 1 FIGURE 1 MICROFERMENTOR ROBERT STEEL INVENTOR ATTORNEYS Jan. 31, 1967 v R. STEEL 3,301,769

MICROFERMENTOR AND PROCESS OF USING Filed Sept. 28, 1964 NOVOBIOCIN, mcg./m|.

3 Sheets-Sheet 2 FIGURE 2 NOVOBIOCIN PRODUCED IN SHAKE FLASK AND MICROFERMENTOR MEDIUM A MEDIUM B NUTRIENT MEDIUM STRENGTH,

' SHAKE FLASK O MICROFERMENTOR ROBERT STEEL INVENTOR 42 may A ORNEYS Jan. 31, 1967 R. STEEL MICROFERMENTOR AND PROCESS OF USING 3 Sheets-Sheet 5 Filed Sept. 28, 1964 mohzuimmmouu U o om o2 V NIOOIHOAON ROBERT STEEL INVENTOR Q/I t;

ATTORNEYS United States Patent 3 301 769 MICROFERMENTOR AND PROCESS OF USING Robert Steel, Kalamazoo, Mich, assignor to The Upjohn Company, Kalamazoo, Mich, a corporation of Delaware Filed Sept. 28, 1964, Ser. No. 399,457 8 Claims. (Cl. 19580) This invention relates to a fermentor and more particularly to a microfermentor for cultivating microorganisms.

The microfermentor of the invention comprises an open-ended rigid cylinder, the ends of which are covered with a membrane permeable to respiratory gases but relatively impermeable to water. Advantageously, the membrane is a polymeric or plastic membrane.

A major effort in fermentation development work concerns studies designed to improve yields of existing fermentation processes. Two of the available procedures used to obtain higher product yields are (1) a study of the influence of nutrients, e.g., sources of carbon, nitrogen, vitamins and growth factors, etc., on microbial growth and yield of desired products, and (2) the testing of mutant strains to find those that produce the desired product in high yield. At present both of these operations are generally carried out in shake flask fermentors. A typical procedure would involve inoculation of 100 ml. of sterile nutrient medium in a 500 ml. conical flask followed by incubation on a shaking machine to provide mixing and to aidoxygen transfer. At the completion of the fermentation the broth is assayed. This overall procedure, because of the size of the flask used, requires extensive shaker capacity and laborious handling and cleaning procedures.

Further, the shaker flasks employed in the above-described procedure are generally stoppered with a porous material, such as cotton, to enable gases to pass in and out of the fermentation flask. It is difficult to gauge accurately the movement of gases through a flask stoppered in such a manner.

The microfermentor of the present invention, which utilizes a gas-to-liquid transfer mechanism, overcomes the problems incurred with the use of a shake flask fermentor.

The microfermentor, described herein, can vary in size to contain from 1 ml. (or even less) to 100 ml. of nutrient medium. Thus, the small size of the microfermentor permits the use of less shaker capacity than that needed for the shake flask fermentor.

The cylinder of the microfermentor can be made of materials such :as glass, plastic, steel, and iron. For economy, and ease of operation, a rigid plastic cylinder is preferred. By using a rigid plastic cylinder, it is possible to discard the fermentor after a single use without incurring much expense.

Permeable membranes which can be employed in the practice of this invention are those that are heat sterilizable, e.g., tetrafluoroethylene, silicone rubber, natural rubher, and polypropylene, as they offer obvious advantages over those that are not heat sterilizable, e.g., polyethylene, although those of the latter group could be sterilized by other means. Other qualities such as being non-toxic, strong, durable, and relatively thin are also desirable. Suitable membranes which can be used are Cohrlastic number 2804 and number 3010, which are manufactured by Connecticut Hard Rubber Company and consist of silicone rubber coated fabric, Teflon which is a trade name for polyfluoroethylene resin manufactured by Dupont, and Pliofilm which is a rubber hydrochloride manufactured by Goodyear Tire and Rubber Company. These are merely exemplary of the permeable membranes which can be utilized in practicing this invention. There are many others which can be readily obtained from the various chemical and rubber manufacturing companies.

fermentation art.

ICC

The use of a polymeric or plastic membrane which is permeable to respiratory gases but relatively impermeable to liquids, such as fermentation medium, is a substantial advance over the cotton stoppers generally used in shake flask fermentors.

Further, the use of a permeable membrane on both ends of the microfermentor cylinder introduces a novel and useful technique in fermentation technology. Heretofore, the use of membranes on both ends of a cylindrical fenmentor has never been considered by those in the The introduction of this novel microfermentor, therefore, holds promise as leading to unheard of procedures for control of gases through a fermentor.

By selection of a suitable membrane material, it is possible to control the oxygen supply rate to the culture at a desired level and/ or to control the release of carbon dioxide from the microfermentor at a desired level. The rate of oxygen transferred to the fermentation medium also can be altered by changing the partial pressure of oxygen in the gas phase surrounding the microfermentor.

The rate of oxygen transfer across the membrane is directly proportion-a1 to (1) the thickness of the unembrane and (2) the partial pressure of oxygen in the gas phase. Accordingly, to obtain a desired oxygen transfer rate, either of these factors can be varied within fairly wide limits. Teflon membrane thickness can vary from 0.25 mil to 5 mil using air at atmospheric pressure as a source of oxygen; or from 1.0 to 25 mil using pure oxygen gas.

The microfermentor of the present invention can be used in any fermentation of microorganisms; this will include the filamentous and nonfilarnentous microorganisms as Well as the anaerobic and aerobic fenmentations.

FIGURE 1 is an elevation of the microfermentor;

FIGURE 2 is a comparison of novobiocin yields in shake flasks and microfermentors;

FIGURE 3 is a comparison of novobiocin yields produced by certain strains of Streptomyces niveous in shake flasks and microfermentor-s.

In FIGURE 1 there is shown a microfermentor which comprises a cylinder 1 having a circular cross-section and mounted with its longitudinal axis in a substantially vertical position. The axial length of said cylinder 1 advantageously being greater than the diameter thereof. The cylinder 1 is sealed at one end with a membrane barrier 2, such as Teflon. An adhesive 3 such as Dow Corning 269 can be used to seal the membrane barrier 2 to the cylinder 1. The opposite end of cylinder 1 is covered with another membrane barrier 4, such as Teflon, held in a retaining ring 5 by means of a gasket 6. Each end of the cylinder 1 is expanded to form a shoulder 7.

The retaining ring 5 is preferably made from a metal such as aluminum which is soft enough to crimp over the shoulder of the cylinder. The gasket 6 can be made of any material, preferably heat sterilizable, which is commonly used in biological wares. Gaskets made of Teflon are used extensively and would be suitable for this purpose.

The following examples are illustrative of the performance of the microfermentor of the invention as compared with a conventional shake flask fermentor. Though these examples illustrate the use of the microfermentor on antibiotic fermentations, this should not be construed'as limiting the application of the microfermentor to such fermentations.

Example 1 To compare the performance of the microfermentor with a conventional shake flask fermentor, novobiocin fermentations were conducted in each using the following procedure: Two different nutrient media (A and B) were prepared. Medium A consisted of the following ingredients:

G./liter Glucose monohydrate 20 Peanut meal Soy protein concentrate 10 The presterilizat-ion pH was 7.0. Medium B consisted of the following nutrients:

G./liter Glucose monohydrate 40 Distillers solubles 40' Tap water q.s. to 1 liter.

Presterilidation pH was adjusted to pH 80 8.2 with 50% sodium hydroxide.

The media were diluted to several different strengths and 100 ml. of each medium (A and B) was dispensed to individual 500 ml. stippled shake flasks. The flasks were closed with a layer of three milk discs and sterilized at p.s.i.-g. steam pressure for min. After cooling, 1 ml. samples were removed aseptically from the shake flasks and added to each of a series of glass microfermentors, using 0.25 mil Teflon as the membranes. The nutrient media in the microfermentors and shake flasks were inoculated by sterile wire loops with a mixture of spores and mycelium of a culture of Streptomyces m'veus, NRRL 2466. The aluminum retaining ring on the top of the microfe-rmentor was then crimped mechanically over the shoulder of the microfermentor. After incubation for 7 days at 28 C. on a rotary shaker operated at 260 r.p.m. with 1% in. radius throw, all fermentations were assayed for novobiocin. The results are shown in FIGURE 2.

Example 2 The ability of different strains of Streptomyces niveus to produce novobiocin in microfermentors and shake flask fermentors was tested. The procedures for sterilization, inoculation, and incubation, were the same as those given in Example 1. The microfermentors were formed from rigid plastic material and the membranes were 0.25 mil Teflon. The fermentation medium used was 10% of iull strength medium B. The results shown in FIGURE 3 show a reasonable correlation between the novobiocin yields obtained in the microfermentors and in shake flask fermentors. The low-yielding strains in the microfierment-or were also low-yielding in shake flasks, and the high-yielding strains in the microfermentors were also high-yielding in shake flasks. These data show that miorofermentors can be used to replace shake flask fermentors for screening strains for novobiocin production.

I claim:

1. A microfermentor for cultivating microorganisms in a liquid nutrient medium which comprises (a) an open-ended rigid solid wall cylinder having a liquid capacity of from 1 to 100 ml., and, (b) a membrane, having a thickness of from 0.25 mil to mil, permeable to respiratory gasses, but relatively impermeable to termentation liquids, covering both ends of said cylinder.

2. A microfermentor, as described in claim I, wherein the open-ended rigid cylinder consists of plastic.

3. A microfer-mentor, as described in claim 1, wherein the open-ended rigid cylinder consists of glass.

4. A microfermentor, as described in claim 1, comprising an open-ended glass cylinder having shoulders formed on each end, a membrane, permeable to respiratory gasses but relatively impermeable to fermentation liquids, sealed across one end, and a second membrane, permeable to respiratory gasses but relatively impermeable to fermentation liquids, disposed across the other end and held in place by retaining means.

5. In a microferrnentor, as described in claim 1, Where the membrane barrier is polyfluoroethylene.

6. A process for the cultivation of microorganisms in a liquid nutrient medium which comprises adding liquid nutrient medium to a rigid cylindrical miorofermentor, which comprises (a) an open-ended rigid solid wall cylinder having a liquid capacity of from 1 to ml., and (b) a membrane, having a thickness of 'from 0.25 mil to 25 mil, permeable to respiratory gasses, but relatively impermeable to fermentation liquids, covering both ends of said cylinder; by aseptically inoculating said medium with a desired microorganism and incubating said microfermentor for a time sufficient to allow the growth of said microorganism.

7. A process for the production of an antibiotic by a fermentation which comprises cultivating the antibioticproducing microorganisms in a rigid cylindrical microfermentor, which comprises (a) an open-ended rigid solid :wall cylinder having a liquid capacity of from 1 to 100 ml., and (b) a membrane, having a thickness of from 0.25 mil to 25 mil, permeable to respiratory gasses, but relatively impermeable to fermentation liquids, covering both ends of said cylinder; permitting the antibiotic to accumulate and assaying for said antibiotic.

8. A process for finding high yielding strains of antibiotic-producing microorganisms which comprises cultivating various microorganism strains in a rigid cylindrica-l microfermentor which comprises (a) an open-ended rigid solid wall cylinder having a liquid capacity of from 1 to 100 ml., and (b) a membrane, having a thickness of from 0.25 mil to 25 mil, permeable to respiratory gasses, but relatively impermeable to fermentation liquids, covering both ends of said cylinder; and determining the presence of said high yielding strains.

References Cited by the Examiner UNITED STATES PATENTS 2,022,820 12/1935 Parkinson et al. 84420 2,620,703 12/1952 Lebensfeld et al. 84420 2,934,989 5/1960 Belli et al. 84-411 X 2,996,429 8/1961 Toulmin --143 X 3,019,685 2/1962 Davis 84-411 3,102,082 8/1963 Brewer 195-439 3,175,944 3/1965 Holksema 19528 X 3,184,395 5/1965 Brewer 195-139 X 3,186,917 6/1955 Gerhardt et al. 19'5-l03.5 X

A. LOUIS MONACELL, Primary Examiner. ALVIN E. TANENHOLTZ, Examiner. 

1. A MICROFERMENTOR FOR CULTIVAING MICROORGANISMS IN A LIQUID NUTRIENT MEDIUM WHICH COMPRISES (A) AN OPEN-ENDED RIGID SOLID WALL CYLINDER HAVING A LIQUID CAPACITY OF FROM 1 TO 100 ML., AND, (B) A MEMBRANE, HAVING A THICKNESS OF FROM 0.25 MIL, PERMEABLE TO RESPIRATORY GASSES, BUT RELATIVELY IMPERMEABLE TO FERMENTATION LIQUIDS, COVEING BOTH ENDS OF SAID CYLINDER.
 7. A PROCESS FOR THE PRODUCTION OF AN ANTIBIOTIC BY A FERMENTATION WHICH COMPRISES CULTIVATING THE ANTIBIOTICPRODUCING MICROORGANISMS IN A RIGID CYLINDRICAL MICROFERMENTOR, WHICH COMPRISES 7A) AN OPEN-ENDED RIGID SOLID WALL CYLINDER HAVING A LIQUID CAPACITY OF FROM 1 TO 100 ML., AND (B) A MEMBRANE, HAVING A THICKNESS OF FROM 0.25 MIL TO 25 MIL, PERMEABLE TO RESPIRTORY GASSES, BUT RELATIVELY IMPERMEABLE TO FERMENTATION LIQUIDS, COVERING BOTH ENDS OF SAID CYLINDER; PERMITTING THE ANTIBOIOTIC TO ACCUMULATE AND ASSAYING FOR SAID ANTIBIOTIC. 